Cell duplication requires copying each organelle during the cell cycle an segregating the copies of each organelle between the daughter cells. We are studying the inheritance of the vacuole (lysosome) of S. cerevisiae, using "vac" mutants which are specifically defective in vacuole inheritance. We have developed an in vitro reaction in which isolated vacuoles form segregation structures (tubules and vesicles) like those seen in intact cells, and these structures lead to vacuole fusion. We have found that in vitro inter-vacuole traffic is defective if the vacuoles and cytosol are prepared from any of several vac mutants. This in vitro reaction requires ATP, several cytosolic components including a novel form of thioredoxin, and membrane components including a small G protein (Ypt7p), NSF (Sec 18p), and alpha-SNAP (Sec 17p). We propose to purify each of the soluble proteins and cofactors, identify and isolate all factors which are peripherally bound to the vacuole, and finally to solubilize the vacuole membrane and reconstitute inheritance- competent proteoliposomes. The functional isolation of the proteins which catalyze vacuole inheritance will allow study of the mechanisms which regulate this process according to the cell cycle and which guide it into the bud (nascent daughter cell) in the intact cell. Organelle inheritance is a new area of cell biology for enzymological dissection, and a detailed picture of this process is essential to our understanding of cell division, development, and differentiation in health and disease.